The present invention relates to mixed lipopeptide micelles for inducing an immune response.
A further object is the use of these micelles for therapeutic purposes.
There are two types of effector immune responses: the humoral response due to antibodies, and the cytotoxic response due to CD8+ T lymphocytes.
An effective cytotoxic response requires the presentation of the antigens to the cytotoxic CD8+ T lymphocytes (CTL), in combination with class I molecules of the Major Histocompatibility Complex (MHC), but also to helper CD4+ T lymphocytes (HTL) in combination with class II MHC molecules.
The use of lipopeptides for inducing a cytotoxic response, in other words the in vivo generation of cytotoxic T lymphocytes, has already been described. In particular, application FR-90 15 870 published under the n° 2 670 787 (Institut Pasteur de Lille, Institut Pasteur, INSERM) discloses lipopeptides composed of a peptide portion comprising 10 to 40 amino acids and a lipid portion which may be derived from fatty acids or steroid groups.
These lipopeptides show a good aptitude for inducing a cytotoxic response. However it was advisable to make them able to induce a better quality response by addition of a helper T response whose importance for effective induction and maintenance of the cytotoxic response is known. It was also advisable to make them able to induce a response in as many individuals as possible.
BOURGAULT et al. (1994, J. Immunol., 2530-2537) induced a CTL and HTL response from a mixture of lipopeptides, in the form of an emulsion with an oily adjuvant.
Nevertheless, it was necessary to add incomplete Freund's adjuvant (IFA). The immunogenicity of the vaccine preparation used necessarily involved the functional co-presentation of the HTL and CTL units located in one or more lipopeptides in the mixture. However, the effectiveness of the co-presentation of the different units involved depended on the combination with the incomplete Freund's adjuvant within a very fine emulsion.
An article under the name of VITIELLO et al. (1995, J. Clin. Invest., 95, 341-349) raised the possibility of inducing a CTL response in a selected human population (HLA-A2) by using a lipopeptide containing a sequential combination of a CTL HLA-A2 antigenic determinant and a multivalent helper (HTL) antigenic determinant. It should be noted that this study was carried out on a genetically restricted population.
This article also reports an experiment during which two types of associations between the HTL antigenic determinant and the CTL antigenic determinant were compared: on the one hand, a covalent sequential combination within the same lipopeptide, and on the other an association by simple mixture of a lipopeptide containing the CTL unit with a peptide containing the HTL unit. The results of this study showed a very clear advantage of the covalent combination compared to the mixture, as performed by the authors, in other words by mixture of solutions containing DMSO and PBS buffer (the peptides or lipopeptides were kept in stock solutions at a concentration of 10-20 mg/ml and diluted with PBS just before use).
However, the combination within the same lipopeptide molecule of the cytotoxic and helper antigenic determinants, although able to induce an effective immune response, required the synthesis of long amino acid sequences presenting the multiple antigenic determinants able to combine with several HLA or superfamilies of class I and class II HLA. The covalent combination of all these units within a single molecule presented technical problems not easily overcome, both from the points of view of synthetic methods and analytical characterisation.
In any case, this article mentions the combination of a lipopeptide and a peptide, and not of two lipopeptides. For this reason no mixed micelle formation could take place.
Another article, published by DON DIAMOND et al. (1997, Blood, 90, n° 5), mentions the immunogenicity of a mixture between a peptide carrying a minimal CTL antigenic determinant (pp 65, sequence 495-503 of the cytomegalovirus matrix protein) and a dipalmitoyl peptide containing an HTL antigenic determinant. The mixture was achieved by mixing solutions in dilute acetic acid or in DMSO, using an ultrasonic treatment for 15 to 30 seconds.
This article thus does not describe a mixture of lipopeptides independently containing a CTL antigenic determinant and an HTL antigenic determinant, but the mixture of a lipopeptide containing an HTL antigenic determinant and a nonapeptide corresponding to a minimal CTL antigenic determinant. In addition, there is no mention of the formation of mixed micelles or of micro-aggregates. In this particular case, however, the possibility of direct combination between the nonapeptide and the class I MHC expressed at the surface of the cells could explain the success of the approach followed. The immunogenicity of the preparation indicates that there was effectively co-presentation of the HTL and CTL antigenic determinants by the same antigen-presenting cell; however, the minimal nonapeptide used has the capacity to link directly with the class I MHC at the surface of the antigen-presenting cell, without its presentation by the cell being necessary.
The authors conclude by recognising that there are still several obstacles to long-term immunity, which confirms the experimental character of this study.
The difficulty of obtaining an immune response depending on a double presentation of the peptides separately presenting the HTL and CTL antigenic determinants is now explained by a publication by STUHLER (1997, Proc. Natl. Acad. Sci. USA, 94, 622-627). To be able to observe the induction of a CTL response, it is absolutely necessary that the HTL and CTL antigenic determinants are present on the surface of the same antigen-presenting cell (APC) to be able to be recognised at the same time by the helper T cells recognising the HTL antigenic determinant and the cytotoxic T cells recognising the CTL antigenic determinant.
It follows from the above that compositions containing within the same micelles, or the same micro-aggregates, on the one hand lipopeptides presenting a CTL antigenic determinant and on the other lipopeptides containing an auxiliary T antigenic determinant, i.e. mixed micelles or micro-aggregates, have never, to the knowledge of the applicant, been described.
However, as described above, it is absolutely necessary that the two antigenic determinants, cytotoxic and helper T, are present on the surface of the same antigen-presenting cell.
In addition to the necessity of a co-presentation of the two antigenic determinants on the surface of the same cell, it is also essential to solubilize the lipopeptides, so as to allow their administration to patients, and their sterilisation by filtration.